CN1568369A - Hybrid interferon/interferon Tau proteins, compositions and methods of use - Google Patents

Abstract

This invention relates to a hybrid interferon fusion protein composed of interferon-α protein, wherein the C-terminal region of the interferon-α protein is replaced by the C-terminal region of interferon-τ. The nucleic acid sequence encoding the interferon fusion protein, expression vectors containing such sequences, and therapeutic applications of the interferon fusion protein are also described. Therapeutic applications include antiviral, anti-cell proliferation, and anti-inflammatory applications. One advantage of the interferon fusion peptide of this invention is its low cytotoxicity when used for therapeutic purposes.

Description

Hybrid interferon/interferon tau proteins, compositions and methods of use

This application claims priority to US Provisional Application No. 60/311,866, filed August 12, 2001, which is hereby incorporated by reference.

field of invention

The present invention relates to hybrid interferon proteins consisting of the C-terminal region of interferon-tau and a region of another interferon.

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Background technique

Interferons (IFNs) are a family of structurally and functionally related proteins that have pleiotropic effects on the growth and function of various cell types. Since their discovery as antiviral agents in 1957, IFNs have been shown to have a variety of potent immunomodulatory effects, including regulation of natural killer cell activity and modulation of major histocompatibility antigen expression, and antiproliferative activity against malignant cells (Walter et al., 1998).

The main types of IFN are IFN-α, -β, -τ and -ω, which are also called type I (acid stable), and IFN-γ is designated type II (acid labile). Table 1 below summarizes the main types of IFNs.

Table 1

Interferon overview Aspect Type I Type I Type I Type II Type α&ω β τ γ Produced by: Leukocytes Fibroblasts Trophoblasts Lymphocytes Antivirus + + + + Antiproliferative + + + + Pregnancy Signs - - + -

The IFN-α family of known proteins consists of at least 14 genes, including a pseudogene and two genes encoding the same protein. Thus, there are 12 individual IFNα proteins produced by 14 genes. The various IFN-α subtypes share approximately 80% identity at the amino acid sequence level.

Interferon alpha-1 (IFNα1), also commonly known as interferon alpha-D (IFNαD), is a widely studied and clinically interesting type I interferon. Recent reports have shown the effectiveness of recombinant IFN[alpha]D in the treatment of various viral diseases in humans and animals (Noisakran and Carr, 2000; Noisakran et al., 1999). Different expression systems have been developed and used for clinical and research purposes on human IFN[alpha]D.

The expression of recombinant human IFNαD (rHulFNαD) was described in 1982 as MaDeP preparation methylotrophic system and E. coli system, both using the lac promoter (De Maeyer, 1982). In 1984, Genentech scientists reported the Saccharomyces cerevisiae system in which the fusion of the IFNαD gene to the α-factor prepro signal sequence produced a secreted IFNαD protein with relatively low biological activity (Singh, 1984). A few years later, in 1989, the secretory expression of the human interferon gene in E. coli and B. subtilis using a streptokinase heterologous expression-secretion signal was developed (Breitling, 1989). In these studies, only the B. subtilis system but not the E. coli system was able to secrete rHulFNaD into the culture medium. A dramatic improvement in the intracellular expression of rHulFNαD in E. coli was reported in 1990, by using a defined medium during fermentation in a batch-reared mode that allowed for more efficient expression in E. coli with a reduced specific growth rate protein. However, there have been no other significant improvements in human IFNαD production over the past 11 years.

The first IFN-τ identified was ovine IFN-τ (OvIFN-τ), a protein of 18-19 kDa. Several isomers were identified in homogenates of concepta (embryos and surrounding membranes) (Martal et al., 1979). Subsequently, the low molecular weight proteins released into the conceptus medium were purified and shown to be heat labile and protease sensitive (Godkin et al., 1982). OvIFN-τ was originally called ovine trophoblast protein-1 (oTP-1) because it is the major secreted protein initially produced by ovine conceptotrophectoderm during the critical period of ovine maternal recognition. Later experiments established that OvIFN-τ is a pregnancy recognition hormone necessary for establishing the physiological response to pregnancy in ruminants such as sheep and cattle (Bazer and Johnson, 1991).

Probing sheep blastocyst banks with synthetic oligonucleotides representing the N-terminal amino acid sequence (Imakawa et al., 1987) yielded IFN-τ cDNA, whose predicted amino acid sequence is similar to that of human, mouse, rat, and porcine IFN-α45 -55% homology, 70% homology with bovine IFN-αll, now called IFN-Ω. It has been reported that several cDNA sequences can exhibit different isoforms (Stewart et al., 1989; Klemann et al., 1990; and Charlier, M. et al., 1991). All of them are about 1 kb, with an open reading frame of 585 bases, encoding a leader sequence of 23 amino acids and a mature protein of 172 amino acids. The predicted structure of IFN-τ is a bundle of 4 helical bundles juxtaposed with amino and carboxy termini, further supporting its classification as a type I IFN (Jarpe et al., 1994).

Although IFN-r exhibits many of the activities typically associated with type I IFNs (see Table 1 above), it differs considerably from other type I IFNs. The most prominent difference is its role in pregnancy, see above. Also different is the viral induction. All type I IFNs, except IFN-τ, are readily induced by viruses and dsRNA (Roberts et al., 1992). Induced IFN-α and IFN-β expression is transient, lasting on the order of hours. In contrast, the synthesis of IFN-τ, once induced, is maintained for several days (Godkin et al., 1982). On a per cell basis, IFN-[tau] is produced over 300-fold more than other type I IFNs (Cross and Roberts, 1991).

Other differences may exist in the regulatory regions of the IFN-r gene. For example, transfection of the human trophoblast cell line JAR with the bovine IFN-τ gene elicited antiviral activity whereas transfection with the bovine IFN-Ω gene did not. This suggests that there are unique processing factors involved in the gene expression of IFN-τ. Consistent with this observation is that although IFN-τ is highly homologous to the proximal promoter region (from 126 to the transcription initiation site) of IFN-α and IFN-β; Enhances the expression of IFN-τ (Cross and Roberts, 1991). Thus, the expression of IFN-τ appears to involve different regulators compared to other type I IFNs.

The expression of IFN-τ may also vary among species. For example, although the expression of IFN-τ in ruminants is restricted to specific stages of conceptus development (mainly at 13-21 days) (Godkin et al. 1982), preliminary studies suggest that human IFN-τ is constitutively Sexual expression (Whaley et al., 1994).

Significantly, the utility of interferons is limited by their toxicity. The use of interferons in the treatment of cancer and viral diseases has caused serious side effects. IFN-[alpha] was introduced for the treatment of chronic hepatitis C in the United States in 1991 and in Japan in 1992 (Saito et al., 2000). However, application of IFN-α doses sufficient to produce clinical efficacy (ie, approximately 1 x ¹⁰⁶ units/therapeutic dose and above) is often accompanied by a "flu-like" syndrome characterized by fever, headache, weakness, arthralgia, and muscle Pain (Tyring et al., 1992). Other toxicities such as nausea, vomiting, diarrhea, and anorexia occurred more frequently at doses of 5-10 x ¹⁰⁶ units/therapeutic and above. Neuropsychiatric symptoms have also been reported with IFN-[alpha] treatment (Dieperink et al., 2000). Furthermore, some studies suggest that the therapeutic efficacy of IFN-α is not dose-dependent (Saito et al., 2000), and that treatment with IFN-α in tumor or viral hepatitis patients is associated with the development and exacerbation of autoimmune disorders (Jimenez-Saenz et al., 2000).

Therefore, there is a need for an interferon protein with high antiviral activity and low cytotoxicity. The present invention seeks to meet these needs.

Summary of the invention

Thus, in one aspect, it is an object of the present invention to provide interferon/interferon-tau hybrid proteins comprising interferon proteins whose C-terminal region is replaced by that of interferon-tau. In one embodiment, the present invention provides a non-tau interferon/interferon-tau hybrid protein, i.e. comprising a non-tau interferon protein, wherein the C-terminal region of the non-tau interferon protein is replaced by an interferon - Substitution of the C-terminal region of tau. In another embodiment, the interferon protein is a type I interferon protein. The type I interferon protein can be interferon-alpha. Preferably, the interferon-alpha is human interferon-alpha. In one embodiment, the human interferon-α is human interferon-αD. In yet another embodiment, the interferon protein is interferon-beta.

In one embodiment, the hybrid protein is capable of exhibiting lower toxicity relative to native interferon, such as interferon-alpha, comprising incubating the first PBMCs in a medium containing at least 2000 antiviral units/ml of the hybrid sample for 7 days; incubate a second sample of PBMCs in medium with the same antiviral units/ml native IFN-α for 7 days; compare the percentage of viable cells remaining in the first and second samples, whereby the higher percentage Viable cells are indicative of relatively low toxicity of IFN species in the culture medium.

In one embodiment of the invention, the interferon protein does not have any substitution of the C-terminal region, but instead the C-terminal region of interferon-tau is added to the full-length interferon protein. Therefore, the interferon protein may have 0 amino acids substituted at the C-terminus. In another embodiment, between about 1-30 amino acids of the C-terminus of the interferon protein are substituted. In yet another embodiment, between about 1-10 amino acids of the interferon protein are substituted at the C-terminus. Preferably, the C-terminus of the last 4 amino acids of the interferon protein are substituted. In a related example, the C-terminal region of human interferon-αD corresponds to a sequence spanning 163-166 residues.

In one embodiment, the hybrid protein includes the C-terminal region of interferon-tau, corresponding to a sequence spanning residues 163-172 of interferon-tau. Preferably, the interferon protein is human interferon-αD, the C-terminal region of human interferon-αD spans residues 163-166, and the C-terminal region of interferon-τ corresponds to residues 163-172 of interferon-τ base sequence.

In one embodiment, the hybrid protein has low cytotoxicity relative to interferon or non-tau interferon protein. In another embodiment, the hybrid protein has increased antiviral activity relative to interferon or non-tau interferon protein. In related embodiments, the hybrid protein has an antiviral activity of at least about 1 x ¹⁰⁸ antiviral units/mg in the MDBK/VSV antiviral system. In another related embodiment, the hybrid protein has an antiviral activity of at least about 2 x ¹⁰⁸ antiviral units/mg in the MDBK/VSV antiviral system.

The present invention contemplates that the interferon-tau protein is ruminant interferon-tau. In one embodiment, the interferon-tau is ovine or bovine interferon-tau. The present invention also contemplates that any of the hybrid proteins described above may be pegylated.

In another aspect, the invention includes nucleic acid molecules encoding any of the hybrid proteins described above. In yet another aspect, the invention includes methods of making interferon hybrid proteins. The steps involved in carrying out the method include placing any of the nucleic acid molecules described above into a recombinant expression system; effecting expression of the nucleic acid molecule to produce a hybrid protein; and recovering the hybrid protein. In one embodiment, the recombinant expression system includes P. pastoris.

In yet another aspect, the present invention includes a pharmaceutical composition comprising the hybrid protein prepared as described above, and a pharmaceutically acceptable carrier. The composition may also contain ribavirin.

In one aspect, the invention also includes a method of inhibiting viral replication in a virus-infected cell. The method comprises contacting the cell with an amount of the hybrid protein effective to inhibit viral replication in the cell.

Methods of inhibiting the growth of tumor cells are also contemplated. The method comprises contacting the tumor cells with an amount of the hybrid protein effective to inhibit their growth.

Another aspect of the invention includes a method of treating an autoimmune disease in a patient. The method comprises administering to a patient any of the hybrid proteins prepared as described above in an amount effective to treat the disease.

Yet another aspect of the invention includes a method of treating chronic inflammation in a patient. The method comprises administering to a patient any of the hybrid proteins prepared as described above in an amount effective to treat inflammation.

Still another aspect of the invention includes a method of treating a disease state responsive to interferon. The method comprises administering to a patient any of the hybrid proteins prepared as described above in an amount effective to treat the condition.

In any of the above methods, administration can be by the following methods of administration: oral, topical, inhalation, nasal and injection.

These and other objects and features of the present invention will be more fully understood from the following detailed description of the invention when read in conjunction with the accompanying drawings.

Brief description of the drawings

Figure 1 shows the oligonucleotide sequence and final ligation product for producing HVV fragment 1;

Figure 2 shows the oligonucleotide sequences and final ligation products used to produce HVV fragment 2;

Figure 3 shows the oligonucleotide sequence and final ligation product for producing HVV fragment 3;

Figure 4 shows the oligonucleotide sequences and final ligation products used to produce HVV fragment 4;

Figures 5A-5C show the sequence and selected restriction sites of the HVV-pPICZ-alpha construct;

Figures 6A-6B show selected HVV protein profiles;

Fig. 7 is the SDS-PAGE gel of P.pastoris HVV recombinant culture supernatant after being induced with 1% methanol for 72 hours;

Figure 8 is an SDS-PAGE gel showing partial purification of HVV protein from P. pastoris supernatant.

Figure 9 is a graphical representation of the percentage of PBMC cell-specific cell death analyzed by flow cytometry.

Figure 10 shows the amino acid sequence alignment of selected interferon proteins.

Detailed description of the invention

I. Definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly used by one of ordinary skill in the art of the invention. Practitioners pay particular attention to the definitions and terminology of the art in Sambrook et al., 1989, and Ausubel FM et al., 1993. It is to be understood that this invention is not limited to the particular methodology, protocols and reagents described, as these may vary.

All publications and patents cited herein are expressly incorporated by reference herein for the purpose of describing and disclosing compositions and methodologies which might be used in connection with the present invention.

A "hybrid" nucleic acid construct or sequence contains a portion of the sequence that is not native to the cell in which it is expressed. With respect to a regulatory sequence, heterozygous means that the regulatory sequence (ie, promoter or enhancer) does not naturally regulate the expression of the same gene it is currently regulating. Typically, hybrid nucleic acid sequences are not endogenous or part of the genome of the cell in which they are present, and are added to the cell by infection, transfection, microinjection, electroporation, or the like. A "hybrid" nucleic acid construct may comprise a regulatory sequence/DNA coding sequence combination that is the same or different than that found in natural cells.

As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. "Expression vector" refers to a vector capable of incorporating and expressing heterologous DNA fragments in heterologous cells. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of suitable expression vectors is within the knowledge of those skilled in the art.

As used herein, an "expression gene cassette" or "expression vector" is a nucleic acid construct produced recombinantly or synthetically by a series of specific nucleic acid elements capable of transcribing a specific nucleic acid in a target cell or in vitro. Recombinant expression cassettes can be inserted into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses or nucleic acid fragments. Typically, the recombinant expression gene cassette portion of the expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter.

As used herein, the term "plasmid" refers to a double-stranded (ds) circular DNA construct used as a cloning vector that forms an extrachromosomal self-replicating genetic element in many bacteria and certain eukaryotic cells.

As used herein, the term "nucleotide sequence encoding a selectable marker" refers to a nucleotide sequence capable of being expressed in a host cell, wherein expression of the selectable marker confers on cells containing the expressed gene in the presence of the corresponding selection agent. ability to grow under conditions.

As used herein, the terms "promoter" and "transcriptional initiator" refer to a nucleic acid sequence that acts to direct the transcription of a downstream gene. The promoter will generally be appropriate to the host cell in which the target gene is to be expressed. A promoter, along with other transcriptional and translational regulatory nucleic acid sequences (also referred to as "regulatory sequences"), is necessary for the expression of a given gene. In general, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, and enhancer or activator sequences.

A "chimeric gene" or "heterologous nucleic acid construct," as defined herein, refers to a non-native gene (ie, that has been introduced into a host) that may consist of distinct gene portions, including regulatory elements. Chimeric gene constructs for transforming host cells typically consist of a transcriptional regulatory region (promoter) operably linked to a heterologous protein coding sequence, or, in the case of a chimeric gene with a selectable marker, operably linked to a selectable marker A gene that encodes a protein that confers antibiotic resistance in transformed host cells. A typical chimeric gene transformed into a host cell of the present invention includes a structural or inducible transcriptional regulatory region, a protein coding sequence and a terminator sequence. If secretion of the target protein is desired, the chimeric gene construct may also include a second DNA sequence encoding a signal peptide.

Nucleic acids are "operably linked" when they are placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader is operably linked to the polypeptide DNA if the polypeptide is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to the coding sequence if they affect the transcription of the sequence or if a ribosome binding site is placed to facilitate translation, it is operably linked to the coding sequence. Generally, "operably linked"means that the DNA sequences being linked are contiguous, and in the case of a secretory leader contiguous and in reading phase. But enhancers don't have to be contiguous. Conjugation is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used by conventional practice.

As used herein, the term "gene" refers to a segment of DNA associated with the production of a polypeptide chain, which may or may not include regions preceding and following the coding region, such as the 5' untranslated region (5'UTR) or "leader" sequences and 3'UTR or "tailing" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

As used herein, "recombinant" with respect to a cell or vector includes modified by the introduction of a heterologous nucleic acid sequence, or derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene in the same form that is not found in the native (non-recombinant) form of the cell, or otherwise the native gene is aberrantly, underexpressed or not expressed at all as a result of human intervention.

The term "introducing" in the context of the insertion of a nucleic acid sequence into a cell means "transfection" or "transformation" or "transduction", including cells involving the insertion of a nucleic acid sequence into a eukaryotic or prokaryotic cell, wherein the nucleic acid sequence can be inserted into the genome of the cell (such as chromosomal, plasmid, plastid, or mitochondrial DNA), conversion to a spontaneous replicon, or transient expression (such as transfected mRNA).

As used herein, the term "expression" refers to the process of producing a polypeptide based on the nucleic acid sequence of a gene. The process includes transcription and translation.

The term "signal sequence" refers to the amino acid sequence of the N-terminal portion of a protein that facilitates secretion of the mature protein from the cell. The mature extracellular protein lacks the signal sequence, which is cleaved during secretion.

The term "host cell" refers to a cell that contains a vector and supports the replication, and/or transcription or transcription and translation (expression) of an expression construct. The host cells used in the present invention may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, plant, insect, amphibian or mammalian cells.

As used herein, the terms "activity" and "biological activity" refer to biological activity associated with a specific target protein, such as enzymatic activity. It is thus concluded that the biological activity of a given protein refers to any biological activity typically ascribed to that protein by those skilled in the art.

"Hepatitis C virus or HCV" refers to the virus species whose pathogenic form causes Non-A Non-B Hepatitis (NANBH), and to the attenuated or defective interfering particles derived therefrom. The HCV genome consists of RNA. RNA-containing viruses have a fairly high spontaneous mutation rate, reported to be on the order of 10 ⁻³ to 10 ⁻⁴ nucleotides per insertion. Since genotypic heterogeneity and mobility are inherent to RNA viruses, there are multiple types/subtypes within the HCV genus, which can be either viral or aviral. The propagation, identification, detection and isolation of various HCV types or episomes are documented in the literature.

"Treating" a condition means administering a therapeutic substance effective to reduce the symptoms and/or lessen the severity of the condition.

"Oral"means any route involving administration via the oral cavity or direct gastric or enteral administration, including gastric administration.

"OAS level"refers to the concentration or activity of 2',5'-oligoadenylate synthase (OAS) protein in the blood.

"Yeast" means ascomycete-forming yeasts (Endosporomycetes), yeasts of the genus Basidiosporium and yeasts belonging to the class Incomplete Fungi (Bacillus). Ascomycetes are divided into two families, Spermophthoraceae and Saccharomycetaceae. The latter consists of four subfamilies, Schizosaccharomyces (eg, Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideac (eg, Pichia, Kluyveromyces, and Saccharomyces). Yeasts of the Basidiosporidium genus include the genera Leupelsporidium, Rhodosporidium, Sporidiobolus, Filamentous Basidiomyces, and Thread-shaped Smut. Yeasts belonging to the class Imperomycetes are divided into two families, Sporobolomycetacea (eg, Holosporomycetacea, Saccharomyces bühlerensis) and Cryptococcaceae (eg, Candida). Of particular interest for the present invention are the following genera: Pichia, Kluyveromyces, Saccharomyces, Schizosaccharomyces and Candida. Of particular interest is P. pastoris. Since the classification of yeast may change in the future, for the purposes of this invention yeast should be limited to that described by Skinner et al. In addition to the foregoing, one of ordinary skill in the art is expected to be familiar with yeast biology and the manipulation of yeast genetics. See, eg, Bacila et al., Rose and Harrison; Strathern et al., incorporated herein by reference.

The nucleotide sequences of the present invention, when operably linked to a yeast promoter, can be used to produce a biologically active mature heterologous protein of interest in a yeast host cell. In this manner, a nucleotide sequence encoding a hybrid precursor polypeptide of the invention is provided in an expression gene cassette introduced into a yeast host cell. These expression cassettes will contain a transcriptional initiation region linked to a nucleotide sequence encoding the hybrid precursor polypeptide. This expression cassette provides multiple restriction sites for the insertion of nucleotide sequences requiring transcriptional regulation of the regulatory regions. The expression gene cassette may additionally contain selectable marker genes.

Cloning or expression vectors may contain additional components, for example, the expression vector may have two replication systems, thus allowing it to operate in two organisms, for example expression in human or insect cells and cloning and amplification in prokaryotic hosts increase.

Both cloning and expression vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Furthermore, for integration of the expression vector, the expression vector contains at least one sequence homologous to the host cell genome, preferably two homologous sequences flanking the expression construct. Integrating vectors can be directed to specific locations in host cells by selecting appropriate homologous sequences to be incorporated into the vector. The construction of integrating vectors is well known in the art.

Cloning and expression vectors will typically contain selectable markers. Typical selectable marker genes encode proteins that can (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, zeocin, or tetracycline, (b) complement auxotrophic deficiencies , or (c) provide key nutrients that cannot be obtained from complex media, for example, the gene encoding bacterial D-alanine racemase.

"IFN-τ" refers to any one of the interferon protein family, which has more than 70%, or preferably more than about 80%, or more preferably more than about 90%, and more preferably More than about 95% amino acid identity, this patent is specifically hereby incorporated by reference in its entirety, or is homologous to the IFN-τ and ovine IFN-τ sequences given in FIG. 10 . Amino acid homology can be determined using, for example, the ALIGN program with default parameters. This program is found in the FASTA Version 1.7 suite of sequence comparison programs (Pearson and Lipman, 1988; Pearson, 1990; programs available from William R. Pearson, Department of Biological Chemistry, Box 440, Jordan Hall, Charlottesville, VA).

"IFN-α" refers to any one of the interferon protein family including the IFN-αD amino acid sequence shown in Figure 10, and refers to proteins with amino acid substitutions and changes, such as neutral amino acid substitutions that do not significantly affect protein activity. Preferably, the sequence comprises the IFN-αD sequence of Figure 10 and has more than about 70%, or preferably more than about 80%, or more preferably more than about 90%, and more preferably more than about 95% amino acid sequence homology thereto sexual protein. Homology can be determined using, for example, the ALIGN program, with default parameters, as described above.

As used herein, the term "IFN expression" refers to the transcription and translation of the above-mentioned IFN gene, and its products include precursor RNA, mRNA, polypeptide, post-translationally processed polypeptide and derivatives thereof. By way of example, detection of IFN expression includes standard cytopathic protection assays. IFN mRNA was detected by Western and Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR).

As used herein, the terms "biological activity of IFN" and "biologically active IFN" refer to any biological activity associated with IFN or any fragment, IFN derivative or analogue, such as enzymatic activity, specifically including Antiviral activity can be detected using standard cytopathic protection assays.

As used herein, the term "modified" form, with respect to an IFN-related protein, refers to a derivative or variant of the native protein. That is, a "modified" form of a protein has a derived polypeptide sequence comprising at least one amino acid substitution, deletion or insertion, with amino acid substitutions being particularly preferred. Amino acid substitutions, insertions or deletions can occur at any residue in the polypeptide sequence and interfere with the biological activity of the protein. Accordingly, the corresponding nucleic acid sequence encoding a variant or derivative protein is considered a "variant" or "modified" form of the gene or coding sequence and is included within the scope of the present invention.

"Variant" means a polypeptide derived from a native polypeptide by: deletion (so-called truncation) or addition of one or more amino acids at the N-terminal and/or C-terminal of the native protein; Deletion or addition of one or more amino acids at multiple positions; or substitution of one or more amino acids at one or more positions of a natural polypeptide. Such variants may arise, for example, from genetic polymorphisms, or from human manipulation. Methods of such manipulations are generally known in the art.

For example, amino acid sequence variants of polypeptides can be prepared by mutation of the cloned DNA sequences encoding any of the hybrid IFNs. Methods of mutagenesis and nucleotide sequence modification are well known in the art. See, eg, Walker and Gaastra, eds. (1983); Kunkel (1985); Kunkel et al. (1987); and Sambrook et al. (1989). Guidance on suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978), incorporated herein by reference. Conservative substitutions, such as exchanging one amino acid for another having similar properties, may be preferred. Examples of conservative substitutions include, but are not limited to, Gly<=>Ala, Val<=>lle<=>Leu, Asp<=>Glu, Lys<=>Arg, Asn<=>GIn, and Phe<=>Trp <=>Tyr.

In constructing variants of heterozygous IFN, modifications can be made such that the variant continues to have the desired activity. Clearly, any mutations made to the DNA encoding the variant protein need not place the sequence out of reading frame, and preferably will not create complementary regions capable of causing secondary mRNA structure.

Thus, the proteins of the invention include hybrid IFN/IFN-T proteins and variants thereof. These variants will be substantially homologous and functionally equivalent to the native protein. A variant of a native protein is "substantially homologous" when its amino acid sequence is at least about 80%, more preferably at least about 90%, most preferably at least about 95% identical to the amino acid sequence of the native protein. Variants may differ by as few as 1, 2, 3 or 4 amino acids. "Functionally equivalent" means that the chain defined by the variant sequence produces a protein having substantially the same biological activity as native IFN. Such functionally equivalent variants containing substantial sequence changes are also encompassed by the present invention. Accordingly, functionally equivalent variants of the hybrid IFN protein would possess sufficient biological activity for therapeutic use. By "therapeutic use" is meant that it is effective in achieving a therapeutic goal, eg protection against encephalitis caused by herpes simplex virus type 1.

Methods for determining functional equivalence are available in the art. Biological activity can be measured using assays specifically designed to detect IFN protein activity, including the assays described herein and in US Patent No. 6,204,022, which is hereby expressly incorporated by reference in its entirety. In addition, antibodies raised against biologically active native proteins can be tested for their ability to bind functionally equivalent variants, efficient binding indicating that the protein has a conformation similar to the native protein.

II. Hybrid interferon fusion protein

The present invention makes some observations about the reduced toxicity caused by the C-terminal region of IFN-τ to support chimeric DNA constructs used to generate A hybrid interferon fusion protein at the N-terminus of an interferon type I polypeptide. Examples of such non-tau interferon type I polypeptides include IFN-beta and various subtypes of IFN-a.

According to Figure 10, such a hybrid interferon fusion protein or polypeptide HW is encoded by a chimeric nucleic acid molecule or produced by a natural chemical ligation reaction, and its N-terminus includes amino acid 1 to amino acid 163 of IFN-αD , the C-terminus includes amino acid 163 to amino acid 172 of IFN-τ. The N-terminal segment contains the N-terminal amino acid sequence of a non-tau interferon polypeptide encoded by the 5' segment of the chimeric nucleic acid molecule. The C-terminal segment contains the C-terminal amino acid sequence of the IFN-τ polypeptide encoded by the 3' segment of the chimeric nucleic acid molecule.

Using peptides corresponding to longer or shorter regions within or extending outside of IFN-τ(163-172) or DNA sequences encoding peptides, in combination with functional assays described herein, antiviral as described in Example 5 Experiments, and the toxicity assay described in Example 6, allow the identification of optimal linkages or amino acid residue positions upstream of which the sequence corresponds to non-τ interferon and downstream of which corresponds to IFN-τ . It is contemplated, for example, that hybrid or chimeric interferons of the invention comprising amino acids 1-166 of human IFN-αD and the 10 last C-terminal amino acids of IFN-τ have low toxicity associated with interferon τ, as well as those associated with IFN-τ. -alpha-related biological activity or biological activity generally considered to be IFN-alpha. For example, IFN-[alpha]/IFN-[tau] hybrids can reduce the toxicity of IFN-[alpha] without interfering with, or even increasing, the antiviral properties of IFN-[alpha].

Preferred embodiments of the invention are fusion proteins wherein the N-terminal fragment is from human IFN-αD and the C-terminal fragment is from sheep IFN-τ. Other suitable IFN sequences are available from GenBank or other public sequence repositories.

As noted above, one very important advantage contemplated of the hybrid interferon fusion protein compositions of the present invention is the reduced toxicity of components associated with natural non-Tau-type interferons, such as components that have been approved as therapeutic agents . The hybrid compositions have the same biological activity as approved non-τI IFNs, with reduced IFN-τ cytotoxicity.

III. Generation of Hybrid Interferon Proteins

In one aspect, the invention includes a method of producing a non-tau interferon/interferon-tau hybrid protein. It has been found that when a P. pastoris expression host is transformed with a vector containing the correct nucleic acid molecule sequence, relatively pure and functionally active non-tau interferon/interferon-tau hybrid protein can be produced in large quantities. Considered below are the steps for implementing the invention.

Sequences, methods and compositions that can be used in the present invention can be found in U.S. Patent Nos. 5,958,402, issued September 28, 1999; 5,942,223, issued August 24, 1999; 5,738,845, issued April 14, 1998; issued on August 17, 2001; 6,204,022 issued on March 20, 2001; 5,906,816 issued on May 25, 1999; 6,060,450 issued on May 9, 2000; and 6,372,206 issued on May 16, 2003; each It is hereby incorporated by reference in its entirety. Additional sequences, methods and compositions that may be used in the present invention can be found in pending U.S. Patent Application Nos. 09/910,406, filed July 19, 2001; and 10/137,127, filed May 2002, each It is hereby incorporated by reference in its entirety.

A. P. pastoris host cells

The choice of host for expression of non-tau interferon/interferon-tau hybrid protein will preferably be yeast. Yeasts useful in the methods of the invention are included within the genus Pichia. Of particular interest is P. pastoris. An example of a P. pastoris strain is X-33.

P. pastoris is able to metabolize methanol by inducing the production of alcohol oxidase since it is its sole carbon source (Cregg, 1993). Most P. pastoris expression strains have one or more auxotrophic mutants that allow selection of expression vectors containing appropriate selectable marker genes during transformation. Prior to transformation, these strains were grown in complex media, but growth in restricted media required the addition of appropriate nutrients. The P. pastoris strains of the present invention include those strains that grow in methanol at wild-type (Mut ⁺ ) rates, as well as those strains that vary in their ability to utilize methanol because of the deletion of one or both AOX genes. Also contemplated are protease-deficient strains, which effectively reduce the degradation of foreign proteins (Brierley, 1998; and W et al., 1995).

The selection of suitable yeast and other microbial hosts for the practice of the present invention is within the purview of those skilled in the art. When selecting a yeast host for expression, suitable hosts include, inter alia, those strains that exhibit good secretion capacity, low proteolytic activity, and good viability. Yeast and other microorganisms are generally available from a number of sources, including Yeast Genetic StockCenter, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); American Type Culture Collection (Manassas.VA); Northern Regional Research Laboratories (Peoria, IL ); and suppliers such as Invitrogen (San Diego, CA).

B. Yeast expression vector

The expression vector used in the present invention contains achimeric gene (or expression gene cassette), designed to be operated in yeast, and there are concomitant sequences upstream and downstream of the expression gene cassette. Companion sequences, which may be of plasmid or viral origin, provide the vector with the necessary properties to enable it to transfer DNA from bacteria to the desired yeast host. Suitable transformation vectors are described below. Suitable components of an expression plasmid, including transcriptional and translational start codons, a signal sequence, a coding sequence for a hybrid IFN, and suitable transcriptional and translational stop codons are also discussed below. An example construct is the pPICZα-HVV plasmid sequence shown in Figures 5A-5C.

i. Transcription & Translation Initiation Codon

The nucleotide sequences of the present invention, when operably linked to a yeast promoter, can be used to produce a biologically active mature heterologous protein of interest in a yeast host cell. In this way, it is provided that the nucleotide sequence encoding the inventive hybrid precursor polypeptide is introduced into a yeast host cell as an expression cassette. These expression cassettes contain a transcriptional initiation region linked to an encoding hybrid precursor polypeptide. Such an expression cassette provides multiple restriction sites for insertion of nucleotide sequences under the transcriptional control of the regulatory regions.

The transcription initiation region, a yeast promoter, provides a binding site for RNA polymerase to initiate downstream (3') translation of a coding sequence. The promoter may be a constitutive or inducible promoter, native or analogous or exogenous or heterologous to a particular yeast host. Additionally, the promoter may be a native sequence or alternatively a synthetic sequence. By exogenous is meant that no transcription initiation region is found in the natural yeast of interest into which the transcription initiation region has been introduced.

Suitable native yeast promoters include, but are not limited to, the wild-type alpha factor promoter, detailed below, and other yeast promoters. Preferred promoters are selected from the list below, including P. pastoris glyceraldehyde 3-phosphate dehydrogenase (GAP), formaldehyde dehydrogenase (FLD1), peroxidase matrix protein (PEX8), and GTP encoding the YPT1 promoter enzyme. A more preferred promoter is the alcohol oxidase AOX1 promoter.

a. AOX1 promoter

Although Pichia encodes two alcohol oxidase genes, AOX1 and AOX2, the AOX1 gene is important for most alcohol oxidase activity in yeast cells (Tschopp et al., 1987; Ellis et al., 1985; and Cregg et al., 1989) . The expression of AOX1 gene is strictly controlled by the level of AOX1 promoter transcription. In cells grown in methanol, approximately 5% of the poly(A) ⁺ RNA was derived from AOX1; however, in cells grown in most other carbon sources, no AOX1 message was detectable (Cregg et al., 1988).

Regulation of the AOX1 gene appears to involve two mechanisms: a repression/derepression mechanism and an induction mechanism, similar to the regulation of the S. cerevisiae GAL1 gene. Unlike regulation of GAL1, the absence of a repressive carbon source, such as glucose in the medium, does not lead to actual transcription of AOX1. The presence of methanol is essential to induce high levels of transcription (Tschopp et al., 1987).

b. GAP promoter

Both Northern and reporter activation results indicated that the P. pastoris GAP gene promoter provided strong constitutive expression in glucose at levels comparable to that observed for the AOX1 promoter (Waterham et al., 1997). The level of GAP promoter activity in cells grown in glycerol and methanol was approximately two-thirds and one-third that of that observed in glucose, respectively. The advantage of using the GAP promoter is that it does not require methanol for induction, nor does it require a culture change from one carbon source to another, allowing for smoother growth of the strain. But because the GAP promoter is constitutively expressed, it might not be a good choice for producing proteins that are toxic to yeast.

c. FLD1 promoter

The FLD1 gene encodes glutathione-dependent formaldehyde dehydrogenase, a key enzyme required for the metabolism of certain methylated amines as a nitrogen source and methanol as a carbon source (Shen et al., 1998). The FLD1 promoter was induced with either methanol as a single carbon source (and ammonium sulfate as a nitrogen source) or methylamine as a single nitrogen source (and glucose as a carbon source). After induction with methanol or methylamine, P _FLD1 was able to express the β-lactamase reporter gene at a level similar to that obtained after induction of the AOX1 promoter with methanol. The FLD1 promoter uses methanol or methylamine, an inexpensive, non-toxic nitrogen source that provides some flexibility for inducing high levels of expression. d. PEX8 and YPT1 promoters

For some P. pastoris strains, the AOX1, GAP, and FLD1 promoters can be very strong and can express genes at very high levels. There is evidence that, for some exogenous genes, high levels of expression from _PAOX1 can overwhelm the cell's post-translational processing, resulting in a significant proportion of the exogenous protein being either unfolded, unprocessed, or unlocalized (Thill et al., 1990 ; Brierley, 1998). For these and other applications, moderately expressing promoters are required. For this target, the P. pastoris PEX8 and YPT1 promoters can be used. The PEX8 gene encodes a peroxidase matrix protein important in peroxidase biosynthesis (Liu et al., 1995). It is expressed at very low but very pronounced levels in glucose and is moderately induced when cells are transferred to methanol. The YPT1 gene encodes a GTPase involved in secretion, and its promoter provides low but substantial expression in media containing glucose, or methanol or mannitol as carbon sources (Sears et al., 1998).

e. Alternative promoters

Synthetic hybrid promoters containing the upstream activator sequence of one yeast promoter that provides inducible expression and the transcriptional activation region of another yeast promoter may also serve as functional promoters in yeast hosts.

Yeast-recognized promoters can also include native non-yeast promoters, which bind yeast RNA polymerase and initiate translation of the coding sequence. Such promoters are available in the art. See, eg, Cohen et al. (1980); Mercereau-Puigalon et al. (1980); Panthier et al. (1980); Henikoff et al. (1981); and Hollenberg et al. (1981), each of which is hereby incorporated by reference literature.

ii. Signal sequence and guide sequence

In addition to encoding the protein of interest, the chimeric gene expressing the gene cassette may encode a signal peptide that processes and translocates the hybrid IFN protein as appropriate.

For the purposes of the present invention, a signal peptide is a precursor sequence, which is the N-terminal sequence of a precursor polypeptide of the mature form of a yeast secreted protein. When the nucleotide sequence encoding the hybrid precursor polypeptide is expressed in a transformed yeast host cell, the signal peptide sequence functions to allow entry of the hybrid precursor polypeptide containing the mature heterologous protein of interest into the endoplasmic reticulum (ER). Movement into the lumen of the endoplasmic reticulum represents the initial step into the secretory pathway of yeast host cells. The inventive signal peptide may be heterologous or native to the yeast host cell. The inventive signal peptide sequence may be a known native signal sequence or any variant thereof which does not adversely affect the signal peptide function as described above.

During entry into the ER, the signal peptide is cleaved to a precursor polypeptide at the site of processing. The processed site contains any peptide sequence that is recognized in vivo by yeast proteolytic enzymes. Such a processing site may be the natural processing site of the signal peptide. More preferably, the natural processing site may be modified, or the processing site may be of synthetic origin, so as to be referred to as a preferred processing site. In terms of the "preferred processing site" to be obtained, the processing site cleaved by yeast proteolytic enzymes in vivo is more effective than the natural site. Examples of preferred processing sites include, but are not limited to, binary peptides, particularly any combination of the two basic residues Lys and Arg, i.e. Lys-Lys, Lys-Arg, Arg-Lys, or Arg-Arg, more preferably Lys-Arg. These sites can be cleaved by the endopeptidase encoded by the P. pastoris KEX2 gene (Julius et al. (1983). As a result, the KEX2 endopeptidase will cleave within the peptide sequence to obtain a mature heterologous protein of interest, which can be Other preferred processing sites are used so that the peptide sequence of interest remains intact (see eg Sambrook et al. (1989)).

A functional signal peptide sequence is very important for the extracellular secretion of heterologous proteins from yeast cells. Additionally, the hybrid precursor polypeptide may contain the secretion leader sequence of a yeast secreted protein to further accelerate this secretion process. When present, the leader peptide is generally positioned immediately 3' to the processing site of the signal peptide sequence. With regard to the "secretion leader sequence" to be obtained, a peptide that directs the movement of a precursor polypeptide, for the purposes of this invention, a hybrid precursor polypeptide containing a mature heterologous protein to be secreted, moves from the ER to the Golgi apparatus, from where it moves into secretory vesicles for secretion across the cell membrane into the cell wall region and/or into the growth medium. The leader peptide sequence may be native or heterologous to the yeast host cell.

The leader peptide sequence of the present invention can be the native sequence of the same yeast secreted protein from which the signal peptide sequence can be derived, the native sequence of a different yeast secreted protein, or a synthetic sequence, or any variant thereof that does not adversely affect the function of the leader peptide .

a. Saccharomyces cerevisiae α-factor prepro peptide

For the purposes of the present invention, the leader peptide sequence, when present, is preferably from the same yeast secreted protein from which the signal peptide sequence was derived, more preferably from the alpha factor protein. A large number of genes encoding the precursor alpha-factor protein have been cloned and their combined signal-leader peptide sequences have been identified. See, eg, Singh et al. (1983). The alpha-factor signal-leader sequence has been used to express heterologous proteins in yeast. See, eg, Elliott et al. (1983); Bitter et al. (1984); and Smith et al. (1985).

α-factors, pheromone-hybridized oligopeptides approximately 11 residues in length, can be produced from larger precursor polypeptides between approximately 100 and 200 residues in length, more typically approximately 120- 160 residues. This precursor (pre) polypeptide contains a signal sequence, approximately 19-23 (more typically 20-22 residues), a leader sequence (pro), approximately 60-66 residues, typically the mature pheromone sequence 2-6 tandem repeat regions. Although the signal peptide sequence and the full-length alpha factor leader peptide sequence can be used, the invention also contemplates shortened alpha factor leader peptide sequences for use with the signal peptide when both elements are present in the hybrid precursor molecule.

Processing of this signal sequence involves three steps. The first step is removal of the pre signal by signal peptidases in the ER. In the second step, Kex2 endopeptidase cuts between Arg-Lys of the pro leader sequence. If present, the Glu-Ala repeat region is rapidly cleaved by the Stel3 protein (Brake et al., 1984).

When the hybrid precursor polypeptide sequence of the invention contains a leader peptide sequence, such as the alpha factor leader sequence, there is a processing site immediately adjacent to the 3' end of the leader peptide sequence. This processing site allows yeast host cell-intrinsic proteolytic enzymes to dissociate from the 5' end of its native N-terminal propeptide sequence, or from the peptide sequence 5' of the mature heterologous hybrid IFN, when the mature heterologous protein of interest is present. The yeast secretory leader sequence was cut off at the end. The processing site may contain any peptide sequence that is recognized in vivo by yeast proteolytic enzymes so that the mature heterologous protein of interest is properly processed. The peptide sequence of this processing site may be the native peptide sequence of the native processing site of the leader peptide sequence. More preferably, the native processing sequence can be modified, or the processing site can be obtained synthetically, so as to be referred to as the preferred processing site as described above.

b. Alternative Signal Sequences

Suitable signal sequences also include P. pastor/s acid phosphatase (PHO1 ) and PHA-E from the phytolectin phaseolin (Cereghino and Cregg, 2000; and Raemaekers et al., 1999). Alternatively, signal sequences may be obtained synthetically or determined from genomic or cDNA libraries using hybridization probe techniques known in the art (see Sambrook et al., (1989).

According to the invention described above, the yeast signal peptide and secretory leader peptide sequences represent portions of the inventive hybrid precursor polypeptide which direct the mature heterologous IFNαD sequence through the secretory pathway of the yeast host cell.

iii. Hybrid IFN peptide coding sequence

Examples of hybrid IFN coding sequences are shown in Table 2-HVV. The starting point of the coding sequence is discussed in Example 1 and illustrated in Figures 1-4.

Where appropriate, the nucleotide sequence encoding the hybrid IFN peptide and any other nucleotide sequence of interest may be optimized for increased expression in transformed yeast. That is, nucleotide sequences can be synthesized using yeast-preferred codons for enhanced expression of these sequences. There are many methods in the art suitable for synthesizing yeast-preferred nucleotide sequences of interest (see, eg, US Patent Nos. 5,219,759 and 5,602,034, which are hereby incorporated by reference in their entirety).

Other sequence modifications are known to enhance expression of nucleotide coding sequences in yeast hosts. These include the elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeat regions and other similar properties detrimental to gene expression. The G-C content of a sequence can be adjusted to an average level for a particular cellular host and can be calculated from a reference of known genes expressed in the host cell. Where possible, the nucleotide coding sequence may be modified to avoid the expected formation of hairpin secondary mRNA structures.

iv. Transcription and translation terminators

The termination regulatory region of the expressed gene cassette may be native to the transcriptional initiation region, or derived from another source, if it is recognized by the yeast host. The termination region may be that of the native alpha factor transcription termination sequence, or another yeast-recognized termination sequence, such as the region of the enzyme mentioned above. Transcription termination regions can be selected, especially for mRNA stability, to enhance expression.

A preferred transcription terminator is the Mat-α (α-factor) transcription terminator. A more preferred transcription termination sequence is the AOX1 transcription termination region as shown in FIG. 1 .

v. Selectable markers

Selectable markers include the biosynthetic pathway gene HIS4 from P. pastoris or S. cerevisiae, ARG4 from S. cerevisiae, and the Sh ble gene from Streptoalloteichus hindustanus, which confer resistance to the bleomycin-related drug Zeocin (Cregg et al., 1985; Cregg and Madden, 1989; and Higgins et al., 1998). A series of recently developed biosynthetic markers include P. pastoris ADE1 (PR-aminoimidazole succinimidazole carboxamide synthetase), ARG4 (arginyl succinate lyase), and URA3 (orotic acid nucleoside 5' -phosphate decarboxylase) gene.

vi. Construction of transformation vector

In the case of heterologous hybrid IFN proteins, each of the other elements present in the hybrid precursor polypeptide may be of known native polypeptide sequence or may be obtained synthetically, including those that do not adversely affect element function as described herein. any of its variants. By "adverse effect" is meant that the presence of a variant form of an element results in a decrease in the biological activity of the secreted mature heterologous protein of interest relative to a hybrid precursor polypeptide containing the native form of the element.

During the preparation of the expression cassette, multiple nucleotide sequence fragments may be manipulated to provide the sequence in the correct orientation and, where appropriate, in the correct reading frame. To this end, linkers or linkers may be used to join fragments of nucleotides or other manipulations involving the provision of convenient restriction sites, removal of redundant nucleotides, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, renaturation, resubstitutions such as transitions and transversions may be involved. See especially Sambrook et al. (1989).

The expression gene cassettes of the invention can be ligated into replicons (eg, plasmids, cosmids, viruses, minichromosomes), thus forming expression vectors capable of self-DNA replication in vivo. A preferred replicon is a plasmid. Such plasmids may be maintained in one or more replication systems, preferably two, to allow for stable survival in prokaryotic host cells for cloning and integration in yeast host cells for expression.

In addition, plasmid expression vectors can be integrated with very high or low plasmid copy numbers. Strains containing multiple integrated copies of an expressed gene cassette can sometimes produce more heterologous protein than single copy strains (Clare et al., 1991).

B. Transformation of Yeast Cells

Yeast cells are transformed with the expression constructs described above, using a variety of standard techniques including, but not limited to, electroporation, microprojectile bombardment, spheroplast production, or whole-cell methods, such as those involving lithium chloride and polyethylene glycol (Cregg et al., 1985; Liu et al., 1992; Waterham et al., 1996; and Cregg and Russell, 1998).

C. Culturing and Obtaining Secreted Hybrid IFN Proteins

Transformants are grown in a suitable nutrient medium and, where appropriate, maintained under selection pressure to ensure retention of endogenous DNA. When expression is inducible, the yeast host is grown to produce a high density of cells, and expression is then induced. Recombinant production of an exemplary hybrid IFN protein, HVV, is described in Example 2. Partial purification of HVV is described in Example 3.

In an important aspect of the present invention, it has been found that transformed P. pastoris is capable of expressing and secreting a hybrid IFN protein with a specific activity as disclosed in Example 4 ranging from about 2.75×10 ⁸ U/mg to about 3× Between 10 ⁸ U/mg. Specific activity can be determined by antiviral assays known to those skilled in the art. An example of an antiviral assay is described in Example 5. Another antiviral assay is described in Pontzer and Johnson, 1985, which is hereby incorporated by reference in its entirety.

D. Isolation of Secreted Human IFNαD

As a secreted protein in Pichia pastoris, one of the main advantages of expressing rHulFNαD is the very low level of contaminating native proteins secreted by Pichia into the medium. The combined use of minimally restricted Pichia medium with a very low protein content resulted in efficient secretion of biologically active rHulFNαD, which constituted the vast majority of the total protein in the medium.

Essentially, the true nature of this secretory expression system is to only allow secretion of the protein into the culture medium as a first step in the purification process. This creates a straightforward and simple procedure for further purification using methods such as molecular sieves and ion exchange chromatography, electrophoresis, dialysis, solvent-solvent extraction, and the like. Such methods are known in the art and described eg in Deutscher, 1990 and Scopes, 1982 .

As described in Example 3, one round of molecular sieve chromatography substantially eliminated high molecular weight contaminating proteins, leaving a very pure hybrid IFN (Figure 8) with very high antiviral activity.

IV. Application

The hybrid IFN proteins of the present invention find use in the treatment of a variety of cancers and viral diseases including those in which other interferons have previously been shown to be active. See, eg, US Patent Nos. 4,885,166, 4,975,276, and 5,939,286, each of which is hereby incorporated by reference in its entirety. An example of a viral disease contemplated for treatment by the present invention is hepatitis C virus (HCV).

HCV is a major public health problem, affecting an estimated 170 million people worldwide, and over 10% of the population in some countries (Lechner et al., 2000). HCV is transmitted essentially by transfusion of infected blood and blood products (Cuthbert, et al., 1994; Mansell et al., 1995). The Centers for Disease Control and Prevention estimates that HCV is responsible for 160,000 new cases of acute hepatitis in the United States each year. Therefore, there is still an urgent medical need for effective anti-HCV drugs.

HCV is a positive-sense, lipid-coated RNA virus of the flavivirus family, approximately 10,000 nucleotides in length (Choo et al., 1989). Unlike hepatitis B virus, HCV has no DNA intermediates and thus cannot be integrated into the host genome (Berenguer et al., 1996). Although HCV has been cloned, nitrogen viruses are still difficult to grow in vitro (Trepo, 2000). HCV is nearly persistent, producing chronic infection in 85% of infected individuals, although the mechanism of its persistence is unknown (Trepo, 2000).

Treatment of HCV focuses primarily on reducing inflammation and hepatocellular damage, thus preventing cirrhosis and hepatocellular carcinoma (Horiike et al., 1998; Benvegnu et al., 1998). Currently available treatments for HCV are effective only on a small subpopulation of infected patients (Magrin et al., 1994; Choo et al., 1991; Choo et al., 1989). IFN-α was introduced for the treatment of chronic hepatitis C in the United States in 1991 and in Japan in 1992 (Saito et al., 2000). However, as noted above, the use of sufficient doses of IFN-a to produce a clinical effect (ie, an amount of approximately 1 x ¹⁰⁶ units/treatment and the amounts described above) is often associated with a number of serious side effects.

The lower cytotoxicity of the hybrid proteins of the invention makes them attractive candidates for the treatment of diseases such as HCV.

V. Pharmaceutical Compositions

The hybrid interferon fusion protein of the present invention can be formulated according to known methods to prepare applicable pharmaceutical compositions. Dosage forms containing interferon or interferon-like compounds have been described previously. Generally, the compositions of the present invention are formulated such that an effective amount of hybrid interferon is combined with a suitable carrier to facilitate effective administration of the composition.

Compositions for use in these treatments are also available in a variety of forms. Examples include solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, liposomes, suppositories, injectable and infusible solutions. The preferred form depends on the desired mode of administration and therapeutic use. The compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants known to those skilled in the art. Preferably, the compositions of the invention may be administered to the patient in unit dosage form, usually one or more times a day.

The hybrid interferon fusion polypeptide or related polypeptides can be administered to patients in any pharmaceutically acceptable form, such as oral intake, inhalation, intranasal spray, intravenous, intramuscular, intralesional or subcutaneous injection. In particular, compositions and methods used for other interferon compounds can be used to deliver these compounds.

But a fundamental advantage of the compounds of the present invention is the extremely low cytotoxicity of the inventive hybrid IFN proteins. Because of their low cytotoxicity, it is possible to use hybrid interferon compositions at concentrations higher than those typically employed with other interferon compounds (eg, IFN[alpha]). Accordingly, it is contemplated that the hybrid interferon compositions of the present invention may be administered at a rate from about 5 x 10 ⁴ to 2 x 10 ⁷ units/day to about 5 x 10 ⁷ units/day, or preferably to about 1 x 10 ⁸ units/day. days, or more preferably to about 1×10 ¹⁰ units/day. In another embodiment, the hybrid interferon composition of the present invention is used at a dosage of about 5×10 ¹¹ units/day, or preferably about 5×10 ¹² units/day.

High doses are preferred for systemic administration. Of course, it should be understood that the compositions and methods of the invention may be used in combination with other therapies, such as ribavirin.

Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-imidazolecarboxamide) is a purine nucleoside analog that has been found to interfere with viral mRNA synthesis and is Inhibits the replication of a wide range of RNA and DNA viruses in vivo and in vitro (Fernandez et al., 1986; Balzarini et al., 1991). Ribavirin has been shown to be effective in normalizing transaminase levels but has little activity on serum HCV RNA titers in chronic hepatitis C patients (DiBisceglie et al., 1992). However, the effective effect of ribavirin is short-lived (Clarke, 2000; Koskinas et al., 1995), and because of its severe side effects, the combination of ribavirin and IFN-α is difficult to tolerate (Cotler et al., 2000). Therefore, the combined application of ribavirin and the hybrid IFN protein of the present invention is superior to the combined application of ribavirin and natural IFN-α.

Once the patient's condition improves, a maintenance dose can be given if necessary. Then, as a function of symptoms, the dose or frequency of administration, or both, can be reduced to a level at which the improved state is maintained. When symptoms are relieved to the desired level, treatment should be discontinued. But the patient needs intermittent therapy based on the symptoms of any disease recurrence for a long time.

From the foregoing it can be found that the various objects and features of the invention have been met.

VI. Table 2

Sequences provided in support of the present invention describe SEQ ID NO. HVV Amino Acid Sequence CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEWRAEIMRSLSLSTNLQERLTKMGGDLNSP HW核苷酸序列TGTGATTTGCCAGAGACTCACTCTTTGGACAACAGAAGAACTTTGATGCTTTTGGCCCAAATGTCTAGAATCTCTCCATCCTCTTGTTTGATGGATAGACACGATTTCGGTTTCCCACAAGAAGAATTTGACGGTAACCAATTCCAAAAGGCTCCTGCTATTTCTGTTTTGCACGAGTTGATTCAACAAATTTTCAACTTGTTCACCACTAAGGACTCTTCTGCTGCCTGGGACGAAGACTTGTTGGACAAGTTCTGTACTGAGCTTTACCAACAATTGAACGACTTGGAGGCTTGTGTTATGCAAGAGGAGAGAGTCGGTGAGACCCCATTGATGAACGCTGATTCCATCTTGGCTGTCAAGAAGTACTTCAGAAGAATTACCTTGTACTTGACCGAAAAGAAGTACTCCCCATGTGCCTGGGAAGTCGTTAGAGCCGAAATCATGAGATCTTT 2 GTCCTTGTCCACTAACTTGCAAGAGAGACTTACCAAGATGGGTGGAGACTTGAACTCTCCATAA

VII. Embodiment

The following examples further illustrate the invention described herein and do not in any way limit the scope of the invention.

Example 1

HVV Cloning Strategy

The HVV molecule is a hybrid molecule, which contains the HuIFNαD molecule, and the last 4 amino acids at the C-terminus are replaced by 10 6C-terminal amino acids from sheep IFN-τ. The resulting amino acid coding sequence was used to generate the corresponding DNA coding sequence whose codon usage has been optimized for expression in Pichia pastoris. Construction of the DNA coding sequence was synthesized by serial cloning of oligonucleotide fragments (approximately 150 base pairs in length; Figures 1-4) into a bacterial vector called G2 (Operon Technologies. Alameda, CA). Each of the four 150 base pair fragments (HVV Fragments 1-4) are created by ligating overlapping synthetic oligonucleotides (<40bp in length) designed with specific restriction enzymes 5' or 3' overhangs to simplify cloning into vectors.

A. Cloning Step #1

Vector to be cut: G2

Cleavage with enzymes: Xbal and BamHI

Fragments connected: Fragment 1 (Figure 1)

Name of the new carrier: G2HVV1 F1

B. Cloning Step #2

Vector to be cut: G2HW1 F1

Cleavage with enzymes: EcoRI and BamHI

Fragments connected: Fragment 2 (Figure 2)

Name of the new carrier: G2HVV1 F2

C. Cloning Step #3

Vector to be cut: G2HVV1 F2

Cleavage with enzymes: Sac I and BamHI

Fragments connected: Fragment 3 (Figure 3)

Name of the new vector: G2HVV1 F3

D. Cloning Step #4

Vector to be cut: G2HVV1 F3

Cleavage with enzymes: EcoRI and BamHI

Fragments connected: Fragment 4 (Figure 4)

Name of the new vector: G2HVV1 F4

E. Final inspection

Cleavage with enzymes: Sac II

The HVV gene was excised from the G2HVV1F4 vector and ligated into the pPICZ-α□ vector (Invitrogen, San Diego) using the Xhol and NotI sites on the plasmid. The sequence of the HVV-pPICZ-α construct is depicted in Figures 5A-5.

Example 2

Recombinant production of HVV in P. pastoris

Selected positive P. pastoris HVV transformants were grown overnight in BMGY medium, then cells were induced in BMMY methanol-containing medium using an _OD600 of 2. Cultures were incubated with 1 ml of 10% methanol at 24 and 48 hours post induction. After 72 hours of culture, whole culture supernatants were harvested, sterile filtered, analyzed by 14% Tris-glycine SDS-PAGE, and stained with colloidal gold Coomassie (Novex, San Diego, CA). Supernatants from pPICZα control plasmid transformants were used as negative controls. The results are shown in Figure 7, showing that clone HVV-6 yielded the strongest expression of HVV.

Example 3

Purification of HVV

Figure 8 illustrates partial purification of HVV protein from P. pastoris supernatant. The culture supernatant (20 ml volume) was buffer exchanged with 10 mM Tris, 150 mM NaCI and concentrated to a final volume of 2 ml (Pre-fraction) before loading onto a HiPrep Sephacryl 26/60S-100 size separation column. The first 120 ml of effluent after injecting the specimen was collected at a rate of 1 ml/min, referred to as fraction 1. Three fractions of 20 ml each were collected and called fractions 2, 3 and 4, respectively. Fractions 2, 3 and 4 were concentrated to 1 and run on a 14% SDS-PAGE gel, stained with Novex colloidal golden blue staining kit.

Example 4

HVV antiviral specific activity

HVV protein was partially purified from HVV clone 16 (Figure 7) P. pastoris recombinant culture supernatant using size separation gel chromatography as described in Example 3. Fraction 3 (Figure 8) from 4 different purification experiments (specimen ID: S-0018; S-0019; S-0063; and S-0064) were each concentrated to 1 ml as described in Example 5 below The total protein concentration and antiviral activity of each specimen were determined. The results are shown in Table 3 below.

table 3

Antiviral Specific Activity of HVV-16 Specimen ID Protein concentration (MG/ML) Antiviral Unit (AVU/ML) Antiviral Specific Activity (AVU/MG) Mean AVU/MG±STD(N) S-0018 0.751 2.22×10 ⁸ 2.96×10 ⁸ 2.86×10 ⁸ ±0.09 (N=4) S-0019 0.696 1.92×10 ⁸ 2.76×10 ⁸ S-0063 1.102 3.11×10 ⁸ 2.82×10 ⁸ S-0064 1.422 4.10×10 ⁸ 2.88×10 ⁸

Example 5

Quantitative Antiviral Test

Madin Darby bovine kidney (MDBK) cells were grown to confluency in 96-well flat bottom culture plates using Eagle's MEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. Once the cells were confluent, the assay medium was Eagle's MEM supplemented with 2% FBS and antibiotics. Challenge virus preparations of VSV were pretested in log serial dilutions to determine the optimal working dilution to obtain 100% CPE on confluent MDBK cells after incubation of virus alone overnight (ie, 16-24 hours). VSV dilutions ranged from 10 ⁻¹ to 10 ⁻⁶ per 50 μl/well, using 4 replicate wells for each dilution, for a final total volume of 200 μl/well in a 96-well plate. _TCID50 was determined based on the dilution of virus that would establish 100% CPE. Now, the seed virus used has 10 ^6.5 TCID ₅₀ /ml. The actual number of infectious virus particles used in the assay is 10 ^3.5 TCID ₅₀ /well.

For interferon detection, MDBK cells were grown to confluence and cells were rinsed once with sterile PBS before the growth medium was removed. Then add three copies of the medium containing interferon or no interferon as a control, use the above-mentioned test medium at 100 μl/well, and use serial 10-fold dilutions (such as 10 ^-1 to 10 ^-10 ). Cells were then incubated at 37°C for 18 hours. Recombinant HuIFNαA (Biosource Intl., Camarillo, CA) can be used as a standard interferon control. After the interferon incubation step, cells were washed twice with PBS before adding 100 μl of the predetermined virus dilution @ 10 ^3.5 TCID ₅₀ /well to each well. Virus was added to interferon-treated assay wells and untreated wells and incubated for an additional 16-24 hours at 37°C. Then 100 μl of medium was removed from each well, replaced with 100 μl of 0.2% neutral red solution (Gibco-BRL), and incubated at 37° C. for 1 hour. All medium was removed and gently rinsed twice with PBS before adding 100 μl of acid alcohol (50% ethanol, 1% acetic acid). The _A550 of the dissolved dye was read with a Bio-Kinetics reader (Bio-Tek Instruments, Winooski VT). The percentage of protection is calculated using the following formula:

1 antiviral unit (U) was defined as 50% protection. Interferon titers (units/ml) will be read as the reciprocal of the dilution (per 0.1 ml specimen) representing 50% protection of the cell monolayer, multiplied by a factor of 10.

Once the antiviral units/ml of interferon were determined using the 10-fold dilution range, this data was used to determine the starting dilution for the two-fold dilution range (maximum 10-fold dilution) of interferon in the assay. This will allow for more accurate antiviral units/ml values for the interferon being tested. Note that this colorimetric method is a modification of the assay published by Crawford-Miksza and (1994).

Example 6

Detection of IFNα analogs in an in vitro toxicity assay in peripheral blood mononuclear cells

The in vitro toxicity of HulFNα, OvIFNτ and IFN hybrid protein can be compared with peripheral blood mononuclear cells (PBMC). The buff superficial fraction of whole blood was diluted 1:4 with PBS and overlaid on Nycoprep 1.077 (Nycomed Pharma, Oslo, Norway). After centrifugation at 600×g at 20°C for 20 minutes, if a band may form on the interface, remove it with a pipette. Then the cells were washed once with PBS, and seeded in a 96-well plate at a concentration of 5×10 ⁵ cells/well. After inoculation, the cells were treated with 2000U/ml to 128,000U/ml of IFNα, IFNτ or IFN hybrid protein, repeated three times. After 12 days of incubation, cells were ready for detection using the Annexin V assay (Pharmingen). Note that these assays are used routinely in laboratories and give us consistent, reliable quantitative results.

Annexin V assay:

Annexin V is a 35-36kD Ca ²⁺ -dependent phospholipid-binding protein with high affinity to phosphatidylserine (PS). In apoptotic cells, the membrane PS is translocated from the inner side of the serosa to the outer leaflet. Annexin V bound cells are undergoing early stages of apoptosis and their membrane PS is exposed. Therefore, staining with Annexin V combined with a viability dye, propylene oxide, can identify early apoptotic cells. This assay does not distinguish between cells that have undergone apoptosis and cells that have died due to necrosis.

PBMCs were harvested from 3 wells and prepared for Annexin V assay as specified in the Pharmingen assay manual. All reagents required for cell staining are supplied with the Annexin V Detection Kit. Stained cells were obtained and analyzed using Becton Dickinson FACScan and CellQuest software. It is important to note that there is some basal level of apoptosis (Annexin V positive, methylene negative) and necrosis (both Annexin V and methylene positive) in the PBMC population, with slight variations in each donor. different. Therefore, untreated cell populations were used to determine basal levels of apoptotic and dead cells. Data for each specimen are expressed as percentage of specific cell death (Annexin V ⁺ or Methylene ⁺ ), calculated from a total of 10,000 cells obtained using the following formula:

*Note: When calculating % specific cell death based on methylene staining, use methylene values in place of Annexin V values.

Example 7

Human PBMCs (5×10 5 /well) were incubated with rHulFN□ or rolFN□ at 1.5× ^{10 5} antiviral U/ml for 12 days. Then double stained with AnnexinV-FlTC and methylene. The percentage of specific cell death was calculated using the following formula: [(% AV+ or PI+ cells in control wells)/(% AV+ or PI+ cells in treated wells)×100].

Table 4

Percentage of specific cell death based on Annexin V positive cells HVV-16 rHulFNαD (Biosource) rHulFNαD (Pepgen) BSA control AVG±STD 12.88±15.44 65.00±22.49 45.46±23.64 7.67±14.57 MEDIAN 4.88 65.78 43.95 0.00 N 6 10 10 10

table 5

Percent specific cell death based on propylene positive cells HVV-16 rHulFNαD (Biosource) rHulFNαD (Pepgen) BSA control AVG±lSTD 2.08±3.85 32.88±15.69 19.32±12.55 3.78±8.19 MEDIAN 0.56 34.59 15.27 0.00 N 6 10 9 10

Example 8

Analysis of the percentage of specific cell death by flow cytometry

Figure 9 shows the percentage of specific cell death analyzed by flow cytometry. Human PBMCs (5×10 ⁵ /well) were incubated with rHulFNα or rolFNτ at 1.5×10 ⁵ antiviral U/ml for 9, 10 and 12 days, and then double-stained with AnnexinV-FITC and methylene. The percentage of specific cell death was calculated using the following formula: [(% AV+ or PI+ cells in control wells)/(% AV+ or PI+ cells in untreated wells) x 100]. Columns and symbols represent the calculated values of Annexin V and methylene, respectively.

All publications and patent applications mentioned in this specification are representative of the level of those skilled in the art to which the invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the present invention has been described in terms of specific embodiments, those skilled in the art will appreciate that various changes and modifications can be made without departing from the spirit of the present invention.

Sequence Listing

<110> Pepgen Corporation

<120> Hybrid Interferon/Interferon Tau Proteins, Compositions and Methods of Use

<130>556008008WO00

<140>Not Yet Assigned

<141>Filed Herewith

<150>US 60/311,866

<151>2001-08-12

<160>2

<170>FastSEQ for Windows Version 4.0

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<211>172

<212>PRT

<213> Artificial sequence

<220>

<223> heterozygous interferon

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Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr Leu Met

1 5 10 15

Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser Cys Leu Met Asp

20 25 30

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45

Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Ile

50 55 60

Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp

65 70 75 80

Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu

85 90 95

Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met

100 105 110

Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr

115 120 125

Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140

Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr Asn Leu Gln Glu

145 150 155 160

Arg Leu Thr Lys Met Gly Gly Asp Leu Asn Ser Pro

165 170

<210>2

<211>519

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<223> heterozygous interferon

<400>2

tgtgatttgc cagagactca ctctttggac aacagaagaa ctttgatgct tttggcccaa 60

atgtctagaa tctctccatc ctcttgtttg atggatagac acgatttcgg tttcccacaa 120

gaagaatttg acggtaacca attccaaaag gctcctgcta tttctgtttt gcacgagttg 180

attcaacaaa ttttcaactt gttcaccact aaggactctt ctgctgcctg ggacgaagac 240

ttgttggaca agttctgtac tgagctttac caacaattga acgacttgga ggcttgtgtt 300

atgcaagagg agagagtcgg tgagacccca ttgatgaacg ctgattccat cttggctgtc 360

aagaagtact tcagaagaat taccttgtac ttgaccgaaa agaagtactc cccatgtgcc 420

tgggaagtcg ttagagccga aatcatgaga tctttgtcct tgtccactaa cttgcaagag 480

agacttacca agatgggtgg agacttgaac tctccataa 519

Claims (27)

1. Interferon, rabbit/interferon hybrid protein comprises:

Interferon protein, the C-end region displacement of the disturbed element-τ of C-end region of wherein said interferon protein,

Wherein, hybrid protein has lower cytotoxicity with respect to interferon protein.

2. the hybrid protein in the claim 1, wherein said interferon protein is an I type interferon protein.

3. the hybrid protein in the claim 2, wherein said I type interferon protein is an interferon-' alpha '.

4. the hybrid protein in the claim 3, wherein said interferon-alpha proteins is a human interferon-alpha.

5. the hybrid protein in the claim 4, wherein said human interferon-alpha is human interferon-alpha D.

6. the hybrid protein in the claim 1, in the wherein said interferon protein approximately the C-end between 0-30 amino acid replaced.

7. the hybrid protein in the claim 6, in the wherein said interferon protein approximately the C-end between 0-10 amino acid replaced.

8. the hybrid protein in the claim 7, last 4 amino acid whose C-ends of wherein said interferon protein are replaced.

9. the hybrid protein in the claim 5, the C-end region of wherein said described human interferon-alpha D is corresponding to the sequence of span 163-166 residue.

10. the hybrid protein in the claim 1, the C-end region of wherein said interferon is corresponding to the sequence of span interferon 163-172 residue.

11. the hybrid protein in the claim 1, wherein said interferon protein is human interferon-alpha D, the described C-end region span 163-166 residue of described human interferon-alpha D, the C-end region of described interferon is corresponding to the sequence of span interferon 163-172 residue.

12. the hybrid protein in the claim 1, wherein said hybrid protein has lower cytotoxicity with respect to interferon protein.

13. the hybrid protein in the claim 1, wherein said hybrid protein have about at least 1 * 10 in the MDBK/VSV antivirus system ⁸The antiviral activity of antiviral unit/mg.

14. the hybrid protein in the claim 1, wherein said I type Interferon, rabbit is an interferon-beta.

15. the hybrid protein in the claim 1, wherein said interferon are sheep or Bov IFN-τ.

16. the hybrid protein in the claim 1, wherein said hybrid protein are long lasting (pegylated).

17. coding is according to the nucleic acid molecule of each hybrid protein among the claim 1-16.

18. make the method for Interferon, rabbit hybrid protein, comprising:

To place recombinant expression system according to the nucleic acid molecule of claim 17;

The expression that influences nucleic acid molecule is to produce hybrid protein; With

Reclaim hybrid protein.

19. the method in the claim 18, wherein said recombinant expression system comprises P.pastoris.

20. a pharmaceutical composition comprises hybrid protein and pharmaceutically acceptable carrier according to claim 1.

21. the composition in the claim 20, wherein said composition further contains virazole.

22. a method that in the cell of infective virus, suppresses virus replication, comprise with cell with suppress viral dosage and contact with effective according to the hybrid protein of claim 1 in time multiplexed cell system.

23. a method that suppresses growth of tumour cell, comprise with cell with contact according to the hybrid protein of claim 1 dosage with effective its growth of inhibition.

24. a method for the treatment of patient's autoimmune disorders comprises that the dosage with effective this disease of treatment gives the patient hybrid protein according to claim 1.

25. a method for the treatment of patient's chronic inflammatory diseases comprises that the dosage with effective treatment inflammation gives the patient hybrid protein according to claim 1.

26. the method for the morbid state that a treatment responds to Interferon, rabbit comprises that the dosage with effective this morbid state of treatment gives the patient hybrid protein according to claim 1.

27. any method among the claim 22-26, wherein said administration is undertaken by following medication: oral, local, suction, intranasal and injection.

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